Abstract
A single amino acid exchange between the major histocompatibility complex molecules HLA-B(*)2705 and HLA-B(*)2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27-restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pK(a) calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation. 1) The interaction of Asp-116 with arginine at peptide position 5, as found in pLMP2 (RRRWRRLTV; viral) and pVIPR (RRKWRRWHL; self), and with lysine in pOmega, as found in gag (KRWIILGLNK; viral), additionally stabilizes the B(*)2705 complexes by approximately 5 and approximately 27 kJ/mol, respectively, in comparison with B(*)2709. 2) The protonation state of the key residues Glu-45 and Glu-63 in the B-pocket, which accommodates pArg-2, affects peptide binding strength in a peptide- and subtype-dependent manner. In B(*)2705/pLMP2, protonation of Glu-45/Glu-63 reduces the interaction energy of pArg-2 by approximately 24 kJ/mol as compared with B(*)2705/pVIPR. B(*)2705/pVIPR is stabilized by a deprotonated Glu-45/Glu-63 pair, evoked by allosteric interactions with pHis-8. The mutual electrostatic interactions of peptide and HLA molecule, including peptide- and subtype-dependent protonation of key residues, modulate complex stability and antigenic features of the respective HLA-B27 subtype.
Highlights
The presentation of peptides by major histocompatibility complex (MHC)4 class I molecules at the cell surface and their recognition by cellular ligands like T cell receptors are fundamental for immune responses
The trimeric MHC class I complex consists of a highly polymorphic transmembrane heavy chain (HC) that is noncovalently associated with a light chain, 2-microglobulin (2m) and a peptidic fragment derived from self or non-self proteins
Spectroscopic Characterization of the HLA Molecules Complexed with Fluorescently Labeled Nonameric Peptides—Purified soluble B*2705 and B*2709 molecules refolded with the different peptides m9, TIS, pVIPR, pVIPR-H8T, pLMP2, pLMP2-T8H, pGR, and gag, labeled with the fluorescence dye LY at positions p6 or p8 via Cys residues that replace the respective peptide amino acids, were characterized by absorption and fluorescence spectroscopy
Summary
This equation involves six fitting parameters, respectively; the fluorescence anisotropy of the folded state (native protein), r0b, and of the unfolded or dissociated state, r0d, the temperature dependence of the anisotropy of the peptide-bound (folded) state, Sb, and of the unfolded or dissociated state, Sd, the enthalpy change, ⌬H0p, and the entropy change, ⌬S0p, for the two-state dissociation reaction, the thermodynamic quantities being determined at the midpoint of the transition. For comparison of the relative complex stability between B*2705 and B*2709 molecules complexed with the same peptide, we calculated the difference in free energy ⌬⌬GB*2709-B*2705, given by Equation 4,. ⌬Gcomplex Ϫ ⌬Gfree was estimated by the sum of the van der Waals interaction energy ⌬GvdW and the Coulomb interaction energy ⌬GCoul between the peptide and the HLA-B27 molecule and the difference in solvation free energies between the complex and the free state as shown in Equation 5. The solvation free energy ⌬Gsolvation is approximated as the sum of the electrostatic solvation (⌬Gelec.solv.) and nonpolar energy (⌬Gnonpolar) contributions [39] The former is computed by numerical solution of the (nonlinear) Poisson-Boltzmann equation using the DELPHI II [35] package, and the latter is approximated to depend linearly on the solvent-accessible surface [39]. Errors for the difference in the binding free energies are estimated to 6 kJ/mol
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